| Background:Helicobacter pylori(H.pylori)is one of the most common pathogenic microorganisms in humans.Its infection can lead to gastritis,peptic ulcer,gastric cancer,and other gastrointestinal diseases.Studies have shown that at least 75% of gastric cancer is closely related to H.pylori infection.Therefore,H.pylori have been listed as a class I carcinogen by the World Health Organization(WHO).H.pylori can secrete a variety of virulence factors in the process of infection,such as urease(Ure),cytotoxinassociated gene A(Cag A),and vacuolating cytotoxin a(VacA),which play an important role in the process of inducing host cell lesions.Studies have confirmed that H.pylori infection can cause mitochondrial dysfunction,apoptosis,and autophagy in gastric mucosal epithelial cells,in which VacA plays an indispensable and key role.However,the molecular mechanism of VacA action and its effect on the pathogenic activity of H.pylori infection are largely unknown or poorly understood.Elucidating the molecular mechanism of the role of virulence factors in the pathogenesis of H.pylori infection is of great significance for the prevention and treatment of gastrointestinal diseases related to H.pylori infection,especially gastric cancer.Objective:To explore the autophagy mechanism of the gastric mucosal epithelial cell damage caused by H.pylori infection,and clarify the molecular mechanism of VacA-induced mitophagy in the process of H.pylori infection.Methods:1)Improve the liquid culture method to facilitate the large-scale culture of H.pylori,and establish a bacterial-cell co-culture system that can prolong the interaction time between H.pylori and host cells.2)Bioinformatics was used to analyze the data set of co-culture(interaction)between H.pylori and gastric mucosal cells,and to find and screen differential genes or signal pathways that may be involved in the pathogenic process of H.pylori;The coculture model of gastric mucosal epithelial GES-1 cells infected by H.pylori was established.The proliferation,apoptosis,migration,invasion,autophagy,and stem changes of GES-1 cells after H.pylori infection were detected by flow cytometry,scratch test,transwell chamber test,and Western blot.3)Recombinant VacA,the mature and active form of VacA(p88),was induced and expressed in vitro by gene recombination technology,and the activity of the vacuolating toxin was measured by a neutral red uptake assay.Flow cytometry,Ed U labeling,immunofluorescence,and Western blot were used to detect the proliferation,apoptosis,mitochondrial membrane potential,and mitophagy of GES-1 cells after p88 treatment.4)The proteins or mitochondrial-related(membrane)proteins interacting with p88 were detected by silver staining,mass spectrometry,immunofluorescence,protease K protection,Co-IP,GST/His pulldown,and Western blot;the protein-protein docking(ZDOCK)method was used to determine the possible interaction sites.Results:1)The improved liquid culture system of H.pylori is more suitable for the growth and proliferation of H.pylori and can culture H.pylori on a large scale in vitro,which can be used for in vitro and in vivo experimental research.At the same time,it was found that an appropriate amount of nickel could promote the proliferation of H.pylori,but higher concentrations showed toxic effects.The established co-culture system of H.pylori and gastric mucosal epithelial cells created a more suitable culture environment for the co-growth of bacteria and cells and prolonged the interaction time between bacteria and cells to a certain extent.Compared with the traditional co-culture model,the survival time of H.pylori was less than 24 h and extended to 96 h,and the infected cells showed obvious vacuolar degeneration.2)Bioinformatics analysis showed that H.pylori infection caused changes in multiple biological pathways such as proliferation apoptosis pathway,metabolic pathway,p53 pathway,and autophagy regulation pathway of gastric mucosal epithelial cells.In the co-culture of H.pylori and gastric mucosal epithelial cells,the differential genes of the short-term(1st generation)effect are concentrated in cytoplasm and mitochondria,which mainly affect cell protein presentation and amino acid biosynthesis;The long-term(30th generations)differential genes mainly focus on the cell membrane and protein and their binding,which affect the processes of nuclear and cytoplasmic transport,cell cycle and mitophagy.The co-culture system of H.pylori and GES-1 cells was established for verification.The experimental results confirmed that H.pylori infection could inhibit the proliferation of GES-1 cells and induce their apoptosis.At the same time,H.pylori infection can significantly promote autophagy in GES-1 cells.Further studies suggest that autophagy induced by H.pylori may be mitophagy.H.Pylori culture filtrate(HPCF)has a similar effect.Chronic infection with H.pylori for a long time induced the proliferation of GES-1 cells,significantly enhanced the level of autophagy,cell migration,and invasion,and significantly enhanced the cell stem and tumorigenicity in vitro.3)VacA(p88)induced in vitro has biological activity and can induce obvious vacuolar degeneration of cells.After p88 treatment,the proliferation ability of GES-1cells decreased,apoptosis increased significantly,mitochondrial membrane potential(MMP)and mitochondrial DNA content of GES-1 cells and He La YFP-Parkin cells decreased,and PINK1/Parkin pathway-dependent mitophagy occurred.By analyzing the molecular mechanisms of p88 inducing mitophagy in host cells,it was confirmed that VacA(p88)binds to cell mitochondrial-related(membrane)proteins Tom20,Tom40,Tom70,STOM,and PGAM5 in various ways.The above results suggest that,in the process of H.pylori infecting gastric mucosal epithelial cells,its secreted VacA is targeted to enter the cell mitochondria,and through the interaction with mitochondrial membrane proteins.It is introduced into the mitochondrial inner membrane to form anion channels,reduce the mitochondrial inner membrane potential and cause mitochondrial dysfunction,promote the accumulation of PINK1 and the recruitment of Parkin to mitochondria,and induce mitophagy,eventually leading to cell damage or death.Conclusion:H.pylori infection induces apoptosis and autophagy of gastric mucosal epithelial cells and enhances the migration,invasion,and stem change of cells.H.pylori virulence factor VacA directly targets mitochondria as a major mediator,causes mitochondrial dysfunction through the interaction with mitochondrial proteins,induces PINK1/Parkin pathway-dependent mitophagy,and then leads to the injury or death of host cells. |
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